PROJECT SUMMARY The Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a devastating cause of acute respiratory failure and results in significant morbidity and mortality in critically ill patients. In this proposal we test the hypothesis that pulmonary endothelial cell (EC) barrier dysfunction in ALI is regulated by the group V phospholipase A2 (gVPLA2). Lung EC dysfunction induced by disease-related stimuli is a critical step in ALI. We previously have identified direct effects of gVPLA2 on lung EC as evidenced by disruption of the EC barrier in vitro. We and others also have demonstrated that gVPLA2 is an important regulator of ALI in vivo that mediates preclinical models in mice, including endotoxin (LPS)- and high tidal volume mechanical ventilation-induced lung injury (VILI). In the current proposal, our novel preliminary data suggest that ALI- relevant stimuli (LPS, mechanical cycle stretch (CS), and pathologic live bacteria (MRSA)) increase expression of the PLA2G5 gene to upregulate gVPLA2 levels in lung EC to mediate EC dysfunction. Therefore, we speculate that gVPLA2 represents a promising therapeutic target for ALI/ARDS. To further characterize the critical role of EC gVPLA2 in ALI, integrated approaches across multiple scientific disciplines will be used to determine the pathobiological function of gVPLA2 in ALI induced by a highly clinically relevant bacterial stimulus?MRSA. SA#1 will determine the mechanisms by which gVPLA2 expression is regulated in pulmonary endothelium in vitro. Regulation of promoter expression for the PLA2G5 gene that encodes gVPLA2 will be determined in lung EC in response to MRSA, including characterization of the effects of disease-associated single nucleotide polymorphisms (SNPs) in the PLA2G5 promoter. Similar studies will determine the epigenetic regulation of expression of the 3?UTR for PLA2G5 by miRNA and SNPs. SA#2 will determine the mechanisms by which gVPLA2 affects pulmonary EC function in vitro. The functional role of gVPLA2 on MRSA-induced lung EC permeability will be extensively characterized using multiple complementary measures of barrier function in vitro, as well as its role in MRSA-induced cytokine production and epigenetic changes. The novel regulation of gVPLA2 expression and function by autophagy will be described. SA#3 will explore the therapeutic potential of gVPLA2 inhibition in acute lung injury induced by MRSA in mice. Studies utilizing gVPLA2 knockout mice, mAb directed against gVPLA2, and pharmacologic inhibition (pre- and post- injury) of gVPLA2 will determine the functional significance of this enzyme in MRSA- induced ALI and the potential therapeutic applications in humans. The contribution of EC-derived gVPLA2 will be determined using a targeted liposomal delivery method to modulate lung EC expression of gVPLA2. Novel epigenetic effects of MRSA will be characterized. Data derived from these proposed studies will elucidate the role of gVPLA2 as a critical mediator in the pathogenesis of ALI and target possible future therapeutic approaches to prevent or reduce vascular dysfunction.